The cornea is a convex transparent barrier that serves to maintain the intact structure of the eye and focus light onto the retina. The cornea derives its structural strength, shape and integrity from corneal collagen. The strength of the intertwined collagen strands is due to covalent cross-links established between and within collagen strands and between collagen and glycoproteins in the matrix. In physiologically robust structure of corneas, the enzyme lysyl oxidase performs the collagen cross-linking in a process called oxidative deamination using molecular oxygen present in the tissue. However, the biomechanical strength of corneal collagen can be reduced by a number of conditions including iatrogenic effect from surgical intervention, prosthesis, or medications, often the cause of corneal weakness can be congenital, idiopathic or due to microbial causes or trauma. In case of corneal weakness, interventional strategies to re-establish, or to improve, the strength of the corneal collagen fibers have to be applied and exploited.
Strengthening the weakened corneal collagen can be performed by chemical, physical and photochemical means. Chemical means such as glutaraldehyde, formaldehyde, glyceraldehyde, ribose, glucose and beta-nitro aliphatic alcohols introduced into the cornea have been investigated. Most methods using such chemical cross-linking agents have been abandoned due to concerns with toxicity and efficacy. Physical methods of strengthening corneal collagen include dehydrothermal treatment (desiccation of water from collagen), thermal heating and UVC or gamma radiation. However, many of these techniques have shown drawbacks including collagen denaturation and degradation of, damaging or killing keratocytes, and potentially toxic side effects.
The most promising method to cross-link compromised corneal collagen is photochemical cross-linking, which has been employed in human clinical use for the treatment of keratoconus and ectasia for some time. This method uses a photosensitizer, usually riboflavin monophosphate, and UVA light to create singlet oxygen in stromal collagen. The singlet oxygen reacts to convert lysine side-chains of collagen fibrils to allysine residues that spontaneously condense to cross link the collagen fibrils. Essentially this mimics the reaction caused by the natural enzyme lysyl oxidase. Photochemical cross-linking of the cornea has been demonstrated to successfully stop and reverse the progression of compromised collagen in keratoconus and ectasia and thousands of patients have received this treatment with few serious adverse events.
Photochemical treatment, sometimes called photodynamic therapy, is affected by three elements 1) excitation light, 2) photosensitizer molecules and 3) molecular oxygen. The goal of the chemical reaction is to produce singlet oxygen and the amount of each of the three variables of the reaction (light, photosensitizer, oxygen) determines the rate and amount of singlet oxygen produced. During the reaction, the molecular oxygen in the tissue is depleted. Available molecular oxygen in the tissue has often been the most limiting aspect of photochemical and photodynamic therapy. When tissue oxygen content is too low, the photochemical reaction produces little singlet oxygen. Instead, the reaction converts water into hydrogen peroxide that is cytotoxic and can stimulate the wound healing response with negative consequences. When the tissue oxygen content is relatively high, the photochemical reaction produces singlet oxygen at a rate approaching 100:1 singlet oxygen over hydrogen peroxide. Therefore, maintaining the molecular oxygen level high is a critical aspect of photochemical cross-linking. A rapid ROS production is important as the amount of oxygen decreases with the progress of time. When the solution warms the oxygen concentration is expected to lower as the oxygen solubility decreases with increasing temperature.
The wavelength of the excitation light determines the absorption features of the photosensitizer. The depth of penetration of the UVA light into the cornea is a function of the absorbance of the photosensitizer at various wavelengths and the concentration and distribution of photosensitizer molecules. This depth of penetration is a critical value in corneal cross-linking because too little penetration gives shallow, perhaps insufficient cross-linking, and too much penetration may injure the endothelium.
For riboflavin the maximum absorbance occurs in the UV light spectrum at around 365 nm and 445 nm. At these wavelengths excitation light is absorbed quickly and penetrates to the least depth into the tissue. The normal cornea is nominally around 500 microns in depth, and a layer of endothelial cells defines the back of the cornea.
Furthermore, the dynamics of the procedure for light penetration and the conversion of molecular oxygen to singlet oxygen depend upon the concentration of the photosensitizer in the tissue and the concentration of oxygen in the tissue.
There are several cautions to be observed when using UVA light and riboflavin for corneal cross-linking.
Firstly, UVA light can have cytotoxic effects on all living cells, and in particular, the corneal endothelial cells on the posterior layer of the cornea can be destroyed by excess UVA light or reactive oxygen species (ROS).
Then, activated riboflavin can produce cytotoxic hydrogen peroxide if oxygen molecules are not available. Such cytotoxic hydrogen peroxide can kill or disable healthy cells. The hydrogen peroxide also acts as a potent chemical messenger to other cells to initiate wound healing responses. These wound healing responses in the cornea may lead to edema, inflammation and differentiation of keratocytes into myofibroblasts with the production of types of collagen not conducive to optically transparent. In fact, corneal haze and scarring can result from myofibroblast formation.
Hence, a quicker reaction assures a less toxic event, and consequently, a better and more efficient action mechanism.
Corneal de-epithelialization is performed to promote riboflavin infusion into the stroma of the eye. The de-epithelialization procedure is intended to ensure that sufficient riboflavin phosphate is introduced into the eye to prevent excessive UVA radiation to the endothelium. In this case, the riboflavin concentration acts as a UVA sunscreen for the deeper endothelium. Additional adverse events may include postoperative infection/ulcer and stromal haze. Some patients report significant discomfort, pain and worse vision lasting roughly a week, and significantly worse vision than prior to the cross-linking procedure lasting for several months. Most of the adverse side effects are a result of the surgical removal of the corneal epithelium prior to the introduction of the riboflavin. The ability to predict the clinical outcome, i.e. the improvement of a patient's best-corrected visual acuity (BCVA) or prevention of reduction of BCVA over the long term, is not high.
In the prior art several alternative compounds to be used for corneal cross-linking in the treatment of keratoconus have been described.
The international patent application WO 2004/058289 discloses ophthalmic compositions comprising EDTA, EDTA sodium, EDTA potassium salt for treating ocular conditions of the cornea such as keratoconus.
Wollensak Gregor et al. in “Collagen cross-linking of human and porcine sclera”, Journal cataract and refractive surgery, 2004, vol. 30, pages 689-695, discloses the process of cross-linking of sclera with glyceraldehyde, glutaraldehyde or riboflavin.
Spoerl E et al. in “Techniques for stiffening the cornea”, J. Refract Surg. 1999, 15: 711-713, discloses corneal cross-linking performed with photosensitizers, such as riboflavin, and chemical cross-linkers, such as glutaraldehyde and Karnovsky's solution.
The application of a photosensitizer such as riboflavin-5-phosphate to a tissue, e.g. cornea, skin, tendon, cartilage, or bone, followed by photoactivation is described in U.S. Pat. No. 7,331,350, producing a tissue-tissue seal to repair a wound or to seal a tissue transplant. Said described method can be applied to different type of surgical procedures such as corneal transplant surgery, cataract surgery, laser surgery, keratoplasty, penetrating keratoplasty, refractive surgery, cornea reshaping.
Patent application US 2008/0015660 describes a method for performing oculoplasty for the treatment of corneal dystrophies/keratoconics including applying a riboflavin solution as photosensitizer to a human eye surface and irradiating the treatment region with controlled photoactivating radiation.
An ocular solution containing approximately 0.05-0.25% w/w of riboflavin phosphate and approximately 20% w/w of dextran for the use in the corneal cross-linking technique for the treatment of keratoconus is the object of the international patent application WO 2009/001396. The innovative contribution of the dextran to this solution is guaranteeing a good muco-adhesiveness to the ocular surface enabling a better performance of the contact and hence of the impregnation of the corneal stroma by the riboflavin solution.
A very simple formulation relating to a collyrium for the treatment of patients suffering from conical cornea has been recently disclosed by the European patent application EP 2 253 321. In such formulation, only containing riboflavin-5-phosphate, sodium chloride, benzal chloride and sterile water, the riboflavin-5-phosphate and the benzal chloride, acting as surface-active agent, assist the penetration of the collyrium in the corneal epithelium; compared to standard collyria for the treatment of conical cornea, the product obtained by this described composition has the advantage of not requiring the removal of the corneal epithelium.
A similar technical solution is obtained through UV-A irradiation of a riboflavin/collagen mixture in the presence of copious oxygen causing rapid cross-linking resulting in adhesion of the mixture in situ effecting its adhesion to underlying ocular structure. Such corneal and sclera tissue seal is disclosed in the International Patent Application WO 2009/073600 in order to obtain a structural augmentation of ocular tissue for better stabilizing progressive corneal diseases.
To overcome the problem of removing the corneal epithelium (de-epithelization), in order to facilitate the riboflavin absorption and the complete imbibition of the corneal stroma before starting the irradiation with UV-A which can create, albeit rarely, complications at a corneal level, pain, in addition to render the task of the oculist more difficult, the international patent application PCT/IT2009/000392, discloses the use of EDTA associated to tromethamine, and/or one or more photoenhancers chosen among: acridine yellow, quinidine yellow, methylene blue, erithrosine, either alone or mixed together, with riboflavin phosphate, for the preparation of ophthalmic compositions for the method of corneal cross-linking in the treatment of the keratoconus or of other ectasic disorders in order to favour the passage of the ophthalmic composition to the stroma through the corneal epithelium. The invention solves the technical problem of the poor capacity of riboflavin for diffusing through the epithelium and hence reaching the corneal stroma. In fact, by the addition of the disclosed association of EDTA and tromethamine, and/or one or more disclosed photoenhancers, the riboflavin phosphate based compound facilitates epithelial absorption associated to corneal CXL, avoiding the resort to the removal of the corneal epithelium, enabling a non-invasive corneal elimination or reduction of the anaesthesia and consequent fast healing without pain or possible complication for the patients.
However, despite the important advances in the relevant field of riboflavin solutions, there is still the need of more efficient ophthalmic compositions released to imbibe corneal stroma in the practice of corneal cross-linking for the treatment of keratoconus.
Therefore, it would be desirable to further improve the absorption of riboflavin, to reduce riboflavin administration time, without requiring the removal of the corneal epithelium, hence obtaining a noninvasive corneal cross-linking with elimination or reduction of the anesthesia, which does not need particular post treatment therapy, no edema due to the removal of the epithelium, and consequent fast healing without pain or possible complications for the patient.
Therefore, the need of alternative formulations of riboflavin composition to be used in corneal cross-linking is strongly felt.
So far, the most and more efficient corneal cross-linking agents employ riboflavin phosphate, that is a polar, negative charged molecule. Such feature does not allow to easily permeate the ocular membranes. A better approach, or at least an improvement of the present state of art, would use riboflavin, that is a lipophilic molecule, much better permeating the ocular membranes, however, the use of such molecule is very tough because this molecule is insoluble in water.
